Team:USP-Brazil/Results:Assemblies

Assemblies and Transformations

To develop the BioBricks characterization, we built the following strains:

Figure 1: Map of strains that will be use for testing the device. DNA construction images hiding RBS and transcription stop sequences.

The P_AOX1 strains are combinations of three DNA elements: the P_AOX1 promoter, the RFP reporter gene and the modified Mxr1 transcription factor (aforementioned on Detecthol). Thanks to Life Technologies, the strain B1 and the Mxr1 modified and minimum were synthesized and this enabled us to construct the others strains.

Comparing the fluorescence time delay and intensity between strains: Control 1; Strain A and strain B1, we will be able to check the strength and the response velocity of the device with modified P_AOX1 and the efficiency of codon-optimization on RFP for P. pastoris. The comparison between control strain 2 with strain B2 will show the P_AOX1 promoter behavior when the modified Mxr1 transcription factor is used. This will also test if the shorter version of Mxr1—consisting in the sequence for the N-terminal 400 amino-acids from Mxr1—cited on literature [1] will work properly.

To assembly those parts we: used the strain B1 as template to built the strain A using PCR (polymerase chain reaction) and restriction enzymes; the control strain 1 was assembled in the pPIC9K plasmid, which was used to transform P. pastoris, allowing to integrate the construction by homologous recombination in the genome; the strains B2 and Control strain 2 will be made by transforming P. pastoris with the corresponding P_AOX1-RFP construction and the Mxr1 plasmid at the same time; the strain C will be built using the RFP condon/otimized and the P_FLD promoter.

We are building our last construction, strain C, and we already have transformed into P. pastoris two of our constructs. The last construction and the other transformations are planned to be done next week. This will allow us to continue the characterization phase, that we have already done for the Control strain 1.

References

[1] PK Parua et al. Pichia pastoris 14-3-3 regulates transcriptional activity of the methanol inducible transcription factor Mxr1 by direct interaction. Molecular Microbology, vol. 85(2): 282-298 (2012).